CONTRIBUTORS OF ORIGINAL ARTICLES IN

VOLUME LX.

New Orleans Medical and Surgical Journal.

VOL. LX.

JULY, 1907.

Original Article.

No. 1

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Methods of Staining and Cultivating the Gonococcus.

By HENRY G. SPOONER, M. D., of Boston.

Like other bacteria, the gonococcus possesses a power of attraction for basic anilin colors, is readily stained by methylen blue, methyl violet, gentian violet, dahlia and fuchsin, but is decolorized with equal facility in alcohol and acids, according to Gram's staining. This ready decolorization is a negative, but valuable diagnostic sign, between the gonococcus and other forms of diplococci, which usually retain a once imbibed staining much more vigorously and are not decolorized, either by alcohol, or acids and particularly by Gram's method. Gram (1) (1884) found that preparations of schizomycetes, when placed in hydrochloric, or nitric acid alcohol were decolored, so that when they were afterwards treated with aqueous solutions of Bismarck-Brown, they had given up their blue color and became bright brown. Bumm (2)

(1885) observed that the gonococcus, when treated by Gram's method, yields its color much more quickly, than the cell elements, as did a series of other diplococci, similar to gonococci, so that he could not consider this characteristic of importance for differential diagnosis. On the contrary, Roux (3) (November 8, 1886) recommended decolorization according to Gram for doubtful cases, for the purpose of differential diagnosis. His method of procedure was as follows: 1. Coloring with an anilin solution of methylen blue; 2, Action of the fluid of Gram (iodin 1 part, iodide of potasium 2 parts, water, 100 parts) during 3 to 4 minutes; 3, Decolorization by alcohol at 90°c; 4, After-staining with an aqueous solution of eosin.

After this series of operations that are not difficult to do, the ordinary microbes of suppuration remain blue upon a rose base, while the gonococci have taken a rose tint, a little more pronounced, than that of the anatomical elements. Allen (4) (1887), Wendt (5) (1887), advocated this method for differential diagnosis, as did Steinschneider and Galewsky (1889). The latter authors stained their cover-glass preparations, according to their modification of Gram's method, namely, laid them in anilin-gentianviolet (Koch-Ehrlich anilin water solution of gentian-violet is prepared as follows: To about 100 c. c. of distilled water, anilin oil is added, drop by drop,' until it has an opaque appearance, the solution being thoroughly shaken after the addition of each drop. It is then filtered into a beaker through moistened filter-paper until the filtrate is perfectly clear. To 100 c. c. of the filtrate add 10 c. c. of absolute alcohol and 11 c. c. of the concentrated alcoholic solution of gentian-violet, or until a metallic lustre appears on the top of the solution) for twenty-five, or thirty minutes, rinsed, placed for five minutes in Gram's solution and then the preparaitons were laid in alcohol, until decolorization, until the fluid dropping from the glass, no longer showed a violet color. Then they were rinsed off, dried and colored afterwards with Bismarck-Brown. The gonococci then have a brown color, all other cocci are black from the combination of the gentian-violet and Bismarck-Brown staining. By this method Steinschneider and Galewsky recognized four kinds of diplococci in the normal urethra, as well as in the secretion of

gonorrhea. Their milk-white and orange yellow diplococci were not decolorized by Gram's method, whereas the gray-white and citron-yellow diplococci were decolorized by this method. As the gray-white diplococcus was found only once in the healthy urethra and the citron-yellow variety not at all, Gram's method gave very accurate results in ruling out any diplococci that may have been present in the normal urethra. Gonococci bore the relation of 4.65 per cent. to the diplocoocci which are decolorized by Gram, therefore the Gram-Roux method, to differentiate gonococci from other diplococci gave entirely sure results in 95.35 per cent. of the

cases.

Hogge() (1893) allowed the preparations of gonorrheal pus fully 25 to 30 seconds in anilin water gentian violet, that he made by addition of 10 parts of a 10 per cent. alcoholic gentian-violet solution to 100 parts of anilin water, treated even so long in iodin potassium iodide solution and then observed after 10 to 15 seconds of action of absolute alcohol, that the gonococci were entirely decolorized. The material used for after-staining was not mentioned. In two observations, a urethritis complicated by a double epididymitis, and one of cystitis, numerous diplococci were found. whose form resembled that of the gonococcus, but he could not distinguish between them by his method of staining according to Gram.

Bröse() (1893) energetically attacked the diagnostic value of microscopical examinations for gonococci, because these germs were absent in many cases, and also because they assumed involution forms. Witte (8) (1893) came to analogous conclusions. Neisser (9) (1885 and 1899) had also called attention to the fact that often secretions must be examined for months in order to find gonococci.

Touton (10) (1894) claimed that many gonococci are not sufficiently or not at all decolorized by Gram's method. Caneva (11) (1894) is of the opinion that gonococci, according to Gram's method, are not decolorized in a certain absolute manner. However, Kral, (1894) under definite quantitative conditions with Gram's method, always obtained positive results.

Kiefer (12) (1895) considered that the concentration of the staining solution had no influence on the prompt decolorization of

the gonococcus, but that rapid decolorization is hindered by too much anilin in the gentian-violet solution, therefore he used only a 2 per cent. anilin water. Van der Bergh (13) (1896) called attention to the fact that the gonococcus situated intracellularly remained constant to Gram's coloring, if the preparations of gonorrheal pus were decolorized in absolute alcohol, not longer than 30 seconds, when the staining solution was not too weak. The same evident uncertainty of Gram's method has been determined by Nogues (14) (1897). Weinrich (15) (1898), upon (1898), upon the basis of his investigations came to the conclusion that the concentration of coloring material and the anilin contents of Ehrlich's gentianviolet solution were not what caused uncertain results of decolorization, but the rinsing of preparations with water, as well as the employment of dilute alcohol.

These different observations can only be explained by the different methods employed by the authors of performing Gram's method, as most of the authors have neglected to state how they have proceeded.

Gram (1884) gave seemingly wide boundaries in relation to the concentration of the color material and the duration of staining. For staining he took the customary Ehrlich's anilin-oil-gentianviolet solution, in which the preparations remained 1 to 3 minutes, then they were conveyed into an aqueous solution of Lugol's solution (iodin 1.0, potassium iodide 2.0, water 300.0), without, or after a slight rinsing with anilin water in which they remained 1 to 3 minutes, then the preparations were placed in absolute alcohol, until they were entirely decolorized, the alcohol being renewed once or twice. Ehrlich's article did not give the time of shaking 5 ccm. anilin oil with 100 ccm. of distilled water, although the solution. must be affected by the length of time and the energy with which the shaking is done. Both of these factors must have an evident influence on the more or less complete blending of the anilin oil in the water. Even so the addition of color material to anilin water, until solution still remains transparent, must give occasion to very different quantitative relations between color material and anilin water, in the hands of different investigators, who use glass vessels of different diameters.

The decoloration of preparations in alcohol to entire decoloration is microscopically scarcely to be controlled.